Silk Fibroin Hydrogels Research Articles

Exosomes derived from hypoxia preconditioned mesenchymal stem cells laden in a silk hydrogel promote cartilage regeneration via the miR-205–5p/PTEN/AKT pathway

Tissue engineering has promising prospects for cartilage regeneration. However, there remains an urgent need to harvest high quality seed cells. Bone marrow mesenchymal cells (BMSCs), and in particular their exosomes, might promote the function of articular chondrocytes (ACs) via paracrine mechanisms. Furthermore, preconditioned BMSCs could provide an enhanced therapeutic effect. BMSCs naturally exist in a relatively hypoxic environment (1%–5% O2); however, they are usually cultured under higher oxygen concentrations (21% O2). Herein, we hypothesized that hypoxia preconditioned exosomes (H-Exos) could improve the quality of ACs and be more conducive to cartilage repair. In our study, we compared the effects of exosomes derived from BMSCs preconditioned with hypoxia and normoxia (N-Exos) on ACs, demonstrating that H-Exos significantly promoted the proliferation, migration, anabolism and anti-inflammation effects of ACs. Furthermore, we confirmed that hypoxia preconditioning upregulated the expression of miR-205–5p in H-Exos, suggesting that ACs were promoted via the miR-205–5p/PTEN/AKT pathway. Finally, an injectable silk fibroin (SF) hydrogel containing ACs and H-Exos (SF/ACs/H-Exos) was utilized to repair cartilage defects and effectively promote cartilage regeneration in vivo. The application of SF/ACs/H-Exos hydrogel in cartilage regeneration therefore has promising prospects. Statement of significanceCartilage tissue engineering (CTE) has presented a promising prospect. However, the quality of seed cells is an important factor affecting the repair efficiency. Our study demonstrates for the first time that the exosomes derived from hypoxia preconditioned BMSCs (H-Exos) effectively promote the proliferation, migration and anabolism of chondrocytes and inhibit inflammation through miR-205–5p/PTEN/AKT pathway. Furthermore, we fabricated an injectable silk fibrion (SF) hydrogel to preserve and sustained release H-Exos. A complex composed of SF hydrogel, H-Exos and chondrocytes can effectively promote the regeneration of cartilage defects. Therefore, this study demonstrates that hypoxia pretreatment could optimize the therapeutic effects of BMSCs-derived exosomes, and the combination of exosomes and SF hydrogel could be a promising therapeutic method for cartilage regeneration.

Muscle-derived stem cells in silk fibroin hydrogels promotes muscle regeneration and angiogenesis in sheep models: an experimental study.

Silk fibroin (SF) hydrogels are of high interest in tissue engineering. However, angiogenesis is one of the major challenges in tissue regeneration and repair. In this study, we present a simple and effective method to develop a 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG)-SF hydrogel. The SF hydrogels had no immunogenicity and approached natural tissues. The SF scaffolds were first prepared from Bombyx mori silkworms and DMPG. The SF scaffold was seeded with muscle-derived stem cells derived from sheep embryo and implanted in the tibialis anterior muscle of mature sheep. Gelation time, H&E staining, and histochemistry were conducted and observed. The suitability of the hydrogels for 3D cell culture was assessed by living cell stain CM-Dil. The results showed that the SF hydrogels resembled the mechanical properties of natural soft tissues better. The results of H&E staining and histochemistry revealed that the degradation rate showed an S-type change, and muscle regeneration and angiogenesis were clearly visible. Adverse effects were not observed in the sheep models. DMPG-induced SF hydrogels can be successfully used for in situ cell encapsulation. It provides promising opportunities in biomedical applications, such as in tissue engineering and regeneration.

Silk-based phyto-hydrogel formulation expedites key events of wound healing in full-thickness skin defect model

Immense socio-economic burden of chronic wound demands effective, low-cost strategies for wound care. Herein, we have developed a chemical crosslinker-free phyto-hydrogel by encapsulating phytochemicals of Aloe vera mucilage extract (AVM) in the self-assembled polymeric chains of two different silk fibroin (SF) proteins (from Bombyx mori and Antheraea assamensis). Additionally, polyvinylpyrrolidone (PVP) has been used as a stabilizer that also contributed to the mucoadhesive property of the composite (SAP; made of SF, AVM, and PVP) hydrogel. The physicochemical properties of the hydrogel were evaluated and compared with SF hydrogel containing only SF proteins without any additives. The biocompatibility assessment of the hydrogel under in vitro conditions has shown improved cellular proliferative and migratory responses, suggesting faster tissue repairability of the hydrogel. A detailed in vivo comparative study with a commercially available DuoDERM® gel revealed that SAP hydrogel not only promoted wound closure but also showed better deposition and remodeling of the extracellular matrix. Moreover, the hydrogel also demonstrated its ability to downregulate pro-inflammatory markers (IL-1β, TNF-α) and upregulation of anti-inflammatory markers (IL-10, TGF-β) at the early stage of healing. Therefore, the bioactive proteins-carbohydrates composite efficiently accelerates skin regeneration and possesses great translational potential to offer a low-cost alternative wound care therapeutic.

GOx/Hb Cascade Oxidized Crosslinking of Silk Fibroin for Tissue-Responsive Wound Repair.

Promising wound dressings can achieve rapid soft-tissue filling while refactoring the biochemical and biophysical microenvironment to recruit endogenous cells, facilitating tissue healing, integration, and regeneration. In this study, a tissue biomolecule-responsive hydrogel matrix, employing natural silk fibroin (SF) as a functional biopolymer and haemoglobin (Hb) as a peroxidase-like biocatalyst, was fabricated through cascade enzymatic crosslinking. The hydrogels possessed mechanical tunability and displayed adjustable gelation times. A tyrosine unit on SF stabilised the structure of Hb during the cascade oxidation process; thus, the immobilized Hb in SF hydrogels exhibited higher biocatalytic efficiency than the free enzyme system, which provided a continuously antioxidative system. The regulation of the dual enzyme ratio endowed the hydrogels with favourable biocompatibility, biodegradability, and adhesion strength. These multifunctional hydrogels provided a three-dimensional porous extracellular matrix-like microenvironment for promoting cell adhesion and proliferation. A rat model with a full-thickness skin defect revealed accelerated wound regeneration via collagen deposition, re-epithelialisation and revascularisation. Enzyme-loaded hydrogels are an attractive and high-safety biofilling material with the potential for wound healing, tissue regeneration, and haemostasis.

Rhein incorporated silk fibroin hydrogels with antibacterial and anti-inflammatory efficacy to promote healing of bacteria-infected burn wounds

Advanced dressings that can simultaneously prevent bacterial colonization/infection and reduce inflammation are highly desired. A simple strategy was developed to incorporate an anti-inflammatory and antibacterial drug rhein into the structure of silk fibroin (SF) matrix to fabricate a hydrogel dressing. The SF/Rhein hydrogels showed fibrous network nanostructure, high water content (~90%), high water adsorption ability (>2 folds of its own weight), acceptable mechanical strength, biocompatibility and antibacterial properties, suitable as dressings for the treatment of bacterial infected wounds. The SF/Rhein hydrogels enhanced the healing rate of burn wounds by reducing inflammation, expediting angiogenesis, and promoting skin appendages formation, being a promising candidate as wound dressings.

Preparation and properties of silk fibroin hydrogel for biological dressing

As a protective layer of a wound, the medical dressing plays an important role in the healing of the wound. The hydrogel dressing is appeared as a new type of medical dressings and has become a research hotspot. Silk fibroin is a natural polymer protein with excellent biocompatibility, mechanical properties, and various plasticity. In this paper, a drug-loaded silk fibroin hydrogel by the polyethylene glycol was coated on cotton fabrics. The obtained biomedical functional textile dressing had antibacterial properties and biocompatibility.

Osteogenic differentiation of encapsulated cells in dexamethasone-loaded phospholipid-induced silk fibroin hydrogels.

The tissue engineering triad comprises the combination of cells, scaffolds and biological factors. Therefore, we prepared cell- and drug-loaded hydrogels using in situ silk fibroin (SF) hydrogels induced by dimyristoyl glycerophosphoglycerol (DMPG). DMPG is reported to induce rapid hydrogel formation by SF, facilitating cell encapsulation in the hydrogel matrix while maintaining high cell viability and proliferative capacity. In addition, DMPG can be used for liposome formulations in entrapping drug molecules. Dexamethasone (Dex) was loaded into the DMPG-induced SF hydrogels together with human osteoblast-like SaOS-2 cells, then the osteogenic differentiation of the entrapped cells was evaluated in vitro and compared to cells cultured under standard conditions. Calcium production by cells cultured in DMPG/Dex-SF hydrogels with Dex-depleted osteogenic medium was equivalent to that of cells cultured in conventional osteogenic medium containing Dex. The extended-release of the entrapped Dex by the hydrogels was able to provide a sufficient drug amount for osteogenic induction. The controlled release of Dex was also advantageous for cell viability even though its dose in the hydrogels was far higher than that in osteogenic medium. The results confirmed the possibility of using DMPG-induced SF hydrogels to enable dual cell and drug encapsulation to fulfil the practical applications of tissue-engineered constructs.

Effects of a plasma jet on electrochemical properties of silk fibroin hydrogel doped with graphene oxide

This study modified hydrogels prepared from silk fibroinand graphene oxideby injecting plasma into the silk. The SF-GO hydrogels, modified by plasma jet with different discharge time (30, 90, and 150 s) and working gases (argon, air, helium), undergo electrochemical AC impedance spectrum tests. After hydrogel modification with the plasma of any working gas, the impedance in the very low frequency (10−2∼100 Hz) decreased with a longer plasma discharge time. In the very low frequency, the sequence of impedance change is Ar group <air group ≈ He group <empty. The electric capacity of the hydrogel showed an increasing trend with those modified by argon plasma and a decreasing trend for those hydrogels modified by air and helium plasma. The sequence of electric capacity change is Ar group >empty >air group >He group. Fourier transform infrared spectrum, and X-ray diffraction spectrum showed a reduction of β-fold structure and graphene oxide content in SF-GO hydrogels modified by plasma.

Self-assembled silk fibroin hydrogels: from preparation to biomedical applications

This review describes the methods to prepare self-assembled silk fibroin hydrogels with a focus on gelation mechanisms and biomedical applications.

Silk Hydrogel Electrostatically Functionalized with a Polycationic Antimicrobial Peptide: Molecular Interactions, Gel Properties, and Antimicrobial Activity.

Functionalization of silk fibroin hydrogel with antimicrobial activity is essential for promoting the applications of this excellent biomaterial. In this work, a simple approach based on electrostatic interaction is adopted to produce antimicrobial silk hydrogel containing an antimicrobial peptide (AMP), polymyxin B, an important last-line antibiotic to treat multidrug-resistant bacterial superbugs. The polycationic property of this peptide and the negative charge of silk fibroin lead to strong interactions between them, as demonstrated by changes in nanofibril structure, gelation kinetics, ζ-potential, fluorescence emission, and rheological properties of the gel. The hydrogels loaded with polymyxin B demonstrated antimicrobial activity against two Gram-negative bacterial strains. A combination of the results from the different characterizations suggests that the optimal molar ratio of polymyxin B to silk fibroin is 1:2.5. As most AMPs are cationic, this electrostatic approach is suitable for the straightforward functionalization of inert silk hydrogel with other AMPs.

Biomaterial-induced conversion of quiescent cardiomyocytes into pacemaker cells in rats.

Pacemaker cells can be differentiated from stem cells or transdifferentiated from quiescent mature cardiac cells via genetic manipulation. Here we show that the exposure of rat quiescent ventricular cardiomyocytes to a silk-fibroin hydrogel activates the direct conversion of the quiescent cardiomyocytes to pacemaker cardiomyocytes by inducing the ectopic expression of the vascular endothelial cell-adhesion glycoprotein cadherin. The silk-fibroin-induced pacemaker cells exhibited functional and morphological features of genuine sinoatrial-node cardiomyocytes in vitro, and pacemaker cells generated via the injection of silk fibroin in the left ventricles of rats functioned as a surrogate in situ sinoatrial node. Biomaterials with suitable surface structure, mechanics and biochemistry could facilitate the scalable production of biological pacemakers for human use.

3D bioprinted silk fibroin hydrogels for tissue engineering.

The development of biocompatible and precisely printable bioink addresses the growing demand for three-dimensional (3D) bioprinting applications in the field of tissue engineering. We developed a methacrylated photocurable silk fibroin (SF) bioink for digital light processing 3D bioprinting to generate structures with high mechanical stability and biocompatibility for tissue engineering applications. Procedure 1 describes the synthesis of photocurable methacrylated SF bioink, which takes 2 weeks to complete. Digital light processing is used to fabricate 3D hydrogels using the bioink (1.5 h), which are characterized in terms of methacrylation, printability, mechanical and rheological properties, and biocompatibility. The physicochemical properties of the bioink can be modulated by varying photopolymerization conditions such as the degree of methacrylation, light intensity, and concentration of the photoinitiator and bioink. The versatile bioink can be used broadly in a range of applications, including nerve tissue engineering through co-polymerization of the bioink with graphene oxide, and for wound healing as a sealant. Procedure 2 outlines how to apply 3D-printed SF hydrogels embedded with chondrocytes and turbinate-derived mesenchymal stem cells in one specific in vivo application, trachea tissue engineering, which takes 2-9 weeks.

Silk fibroin hydrogels induced and reinforced by acidic calcium phosphate – A simple way of producing bioactive and drug-loadable composites for biomedical applications

Silk fibroin (SF) hydrogels have attracted extensive interest in biomedical applications due to their biocompatibility and wide availability. However, their generally poor mechanical properties limit their utility. Here, injectable, ready-to-use SF-based composites, simultaneously induced and reinforced by acidic calcium phosphates, were prepared via a dual-paste system requiring no complex chemical/physical treatment. The composite was formed by mixing a monocalcium phosphate monohydrate paste with a β-tricalcium phosphate/SF paste. The conformational transition of SF in an acidic environment forms continuous networks, and the acidic calcium phosphate, brushite and monetite, formed simultaneously in the networks during mixing. The composites displayed a partly elastomeric compression behavior, with mechanical properties increasing with an increasing calcium phosphate and β-sheet content at the lower calcium phosphate contents evaluated (22.2–36.4 wt%). While the stiffness was still relatively low, the materials presented a high elasticity and ductility, and no failure at stresses in the range of failure stresses of trabecular bone. Furthermore, the calcium phosphate confers bioactivity to the material, and the composites with a promising in vitro cell response also showed potential as drug vehicles, using vancomycin as a model drug. These dual-paste systems exhibit potential utility in biomedical applications, such as bone void fillers and drug vehicles.

Computational analysis of vincristine loaded silk fibroin hydrogel for sustained drug delivery applications: Multiphysics modeling and experiments

In this paper, silk fibroin hydrogel is used as a drug carrier for vincristine. To optimize drug delivery, a multi-physics model is proposed that couples the deformation and diffusion fields. We applied inverse analysis and general continuum mechanics to define material parameters and mechanical properties. To examine the mass transport and chemical behavior, an affinity-based diffusion and degradation of a drug-loaded polymer matrix is employed. Some experiments are carried out to examine the capability of the presented model. After preparing the vincristine loaded silk hydrogel syringes, they were injected into PBS and enzyme solutions to monitor the drug release rate for 40 days. Obtained results from the computational simulation and laboratory tests showed that the silk fibroin hydrogel was deswelled after about 40 days in enzyme solution. Degradation led to faster and higher doses of vincristine drug release in comparison to the case of PBS solution. Results revealed that more than 80% of the drug was released in the first 5 days in the enzyme solution, but in PBS solution only 10% of the drug was released during 40 days. The model predictions of deswelling behavior and drug release rate were in good agreement with those of experimental results. Therefore, it can be employed as a reliable tool for further predictions.

Methacrylated Silk Fibroin Hydrogels: pH as a Tool to Control Functionality.

The last decade has witnessed significant progress in the development of photosensitive polymers for in situ polymerization and 3D printing applications. Light-mediated sol-gel transitions have immense potential for tissue engineering applications as cell-laden materials can be crosslinked within minutes under mild environmental conditions. Silk fibroin (SF) is extensively explored in regenerative medicine applications due to its ease of modification and exceptional mechanical properties along with cytocompatibility. To efficiently design SF materials, the in vivo assembly of SF proteins must be considered. During SF biosynthesis, changes in pH, water content, and metal ion concentrations throughout the silkworm gland divisions drive the transition from liquid silk to its fiber form. Herein, we study the effect of the glycidyl-methacrylate-modified SF (SilkMA) solution pH on the properties and secondary structure of SilkMA hydrogels by testing formulations prepared at pH 5, 7, and 8. Our results demonstrate an influence of the prepolymer solution pH on the hydrogel rheological properties, compressive modulus, optical transmittance, and network swellability. The hydrogel pH did not affect the in vitro viability and morphology of human dermal fibroblasts. This work demonstrates the utility of the solution pH to tailor the SilkMA conformational structure development toward utility and function and shows the need to strictly control the pH to reduce batch-to-batch variability and ensure reproducibility.

Synergic adhesive chemistry-based fabrication of BMP-2 immobilized silk fibroin hydrogel functionalized with hybrid nanomaterial to augment osteogenic differentiation of rBMSCs for bone defect repair

Bone defect repair and tissue engineering is specifically challenging process because of the distinctive morphological and structural behaviours of natural bone with complex healing and biochemical mechanisms. In the present investigation, we designed dopamine adhesive chemistry-based fabrication of silk fibroin hydrogel (SFD) with incorporation of nano-hydroxyapatite (nHA)-graphene oxide (GO) hybrid nanofillers with well-arranged porous morphology immobilized with bone morphogenic protein-2 (BMP-2) for the effective in vitro rabbit bone marrow derived mesenchymal stem cells loading compatibility and in vivo new bone regrowth and collagen deposition ability. We have achieved bone-specific hydrogel scaffolds with upgraded structural features, mechanical properties and particularly promoted in vitro osteogenic differentiation and compatibility of rabbit bone marrow mesenchymal stem cells (rBMSCs). Structural and microscopic analyses established greater distributions of components and well-ordered and aligned porous structure of the hydrogel network. In vivo result of new bone regrowth was promisingly higher in the Bm@nHG-SFD hydrogel (85%) group as compared to the other treatment groups of nHG-SFD (77%) and nH-SFD (64%) hydrogel. Overall, we summarized that morphologically improved hydrogel material with immobilization of BMP-2 could be have more attentions for new generation bone regeneration therapies.

Progress in the application of silk fibroin in tissue engineered drug delivery system

The properties and characteristics of different types of silk fibroin (SF) drug-loaded sustained-release carriers and their effects on the drug release behavior were reviewed, and the existing problems and development prospects of SF drug-loaded sustained-release carriers in tissue engineering drug delivery system were discussed. The literatures about drug-loaded SF sustained-release carriers in recent years were extensively consulted, and the types of sustained-release carriers, characteristics of drug release, range of applications, advantages and disadvantages, and solutions were summarized and analyzed. At present, the SF drug-loaded sustained-release carriers are mainly divided into SF microparticles, SF scaffolds, SF membranes, SF hydrogels, SF nanofibers, SF sponges, and so on. These types of SF drug-loaded sustained-release carriers have their own advantages and problems, of which the most prominent problem is the burst release of drugs at the initial stage. While, the initial burst release of drugs can be effectively solved by improving the preparation process and adjusting the material ratio. Different types of drug-loaded sustained-release carriers can be prepared by combining different materials to achieve different application scopes and drug release behaviors under different conditions. SF is a good drug-loaded carrier for tissue engineering, the burst release of drugs at the initial stage can be solved by improving the preparation process and changing the material structure; through the combination of the advantages of various types of SF drug-loaded sustained-release carriers, it is expected to prepare SF drug-loaded sustained-release carriers that meet different clinical needs.

Porcine skeletal muscle tissue fabrication for cultured meat production using three-dimensional bioprinting technology

Cultured meat produced through in vitro cell culture technology is regarded as a technical revolution. In this study, three-dimensional (3D) bioprinting technology was used to mimic the growth environment in vivo and construct a 3D culture system in vitro . Hydrogel bioinks, namely, sodium alginate-gelatin and gelatin-methacrylate (GelMA)–silk fibroin, produced using two different curing processes were blended, and their rheological properties, mechanical properties, and biocompatibilities were compared. The 4% GelMA‒20% silk fibroin hydrogel (GS2) demonstrated good performance and was hybridized with porcine skeletal muscle satellite cells for 3D printing to construct network structures of size 15 mm × 15 mm and porosity 1 000 μm in 4-, 6-, and 8-layer structures. After 16 days of culture, 4- and 6-layer grid structures formed compact muscle fibers organized by multinucleated myotubes. These results suggested that 3D bioprinting and GelMA-silk fibroin hydrogels have great potential in fabricating porcine skeletal muscle tissue for use as cultured meat.

Novel fabrication of antibiotic containing multifunctional silk fibroin injectable hydrogel dressing to enhance bactericidal action and wound healing efficiency on burn wound: In vitro and in vivo evaluations.

The development of biologically active multifunctional hydrogel wound dressings can assist effectively to wound regeneration and also has influenced multiple functions on wound injury. Herein, we designed a carbon‐based composited injectable silk fibroin hydrogel as multifunctional wound dressing to provide effective anti‐bacterial, cell compatibility and in vivo wound closure actions. Importantly, the fabricated injectable hydrogel exhibit sustained drug delivery properties, anti‐oxidant and self‐healing abilities, which confirm that composition of hydrogel is highly beneficial to tissue adhesions and burn wound regeneration ability. Frequently, designed injectable hydrogel can be injected into deep and irregular burn wound sites and would provide rapid self‐healing and protection from infection environment with thoroughly filled wound area. Meanwhile, incorporated carbon nanofillers improve injectable hydrogel strength and also offer high fluid uptake to hydrogel when applied on the wound sites. In vitro MTT cytotoxicity assay on human fibroblast cell lines establish outstanding cytocompatibility of the injectable hydrogel and also have capability to support cell growth and proliferations. In vivo burn wound animal model results demonstrate that the hydrogel dressings predominantly influenced enhanced wound contraction and also promoted greater collagen deposition, granulation tissue thickness and vascularization. This investigation's outcome could open a new pathway to fabricate multifunctional biopolymeric hydrogel for quicker burn wound therapy and effectively prevents microenvironment bacterial infections.

Human Umbilical Cord Mesenchymal Stem Cell Derived Exosomes Delivered Using Silk Fibroin and Sericin Composite Hydrogel Promote Wound Healing.

Recent studies have shown that the hydrogels formed by composite biomaterials are better choice than hydrogels formed by single biomaterial for tissue repair. We explored the feasibility of the composite hydrogel formed by silk fibroin (SF) and silk sericin (SS) in tissue repair for the excellent mechanical properties of SF, and cell adhesion and biocompatible properties of SS. In our study, the SF SS hydrogel was formed by SF and SS protein with separate extraction method (LiBr dissolution for SF and hot alkaline water dissolution for SS), while SF-SS hydrogel was formed by SF and SS protein using simultaneous extraction method (LiBr dissolution for SF and SS protein). The effects of the two composite hydrogels on the release of inflammatory cytokines from macrophages and the wound were analyzed. Moreover, two hydrogels were used to encapsulate and deliver human umbilical cord mesenchymal stem cell derived exosomes (UMSC-Exo). Both SF SS and SF-SS hydrogels promoted wound healing, angiogenesis, and reduced inflammation and TNF-α secretion by macrophages. These beneficial effects were more significant in the experimental group treated by UMSC-Exo encapsulated in SF-SS hydrogel. Our study found that SF-SS hydrogel could be used as an excellent alternative to deliver exosomes for tissue repair.